Differentiation of Human Embryonic Stem Cells

ABSTRACT

The present invention provides methods to promote the differentiation of pluripotent stem cells into insulin producing cells. In particular, the present invention provides a method to produce a population of cells, wherein greater than 85% of the cells in the population express markers characteristic of the definitive endoderm lineage.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/378,472, filed Aug. 31, 2010, which isincorporated herein by reference in its entirety for all purpose.

FIELD OF THE INVENTION

The present invention provides methods to promote the differentiation ofpluripotent stem cells into insulin producing cells. In particular, thepresent invention provides a method to produce a population of cells,wherein greater than 85% of the cells in the population express markerscharacteristic of the definitive endoderm lineage.

BACKGROUND

Advances in cell-replacement therapy for Type I diabetes mellitus and ashortage of transplantable islets of Langerhans have focused interest ondeveloping sources of insulin-producing cells, or β cells, appropriatefor engraftment. One approach is the generation of functional β cellsfrom pluripotent stem cells, such as, for example, embryonic stem cells.

In vertebrate embryonic development, a pluripotent cell gives rise to agroup of cells comprising three germ layers (ectoderm, mesoderm, andendoderm) in a process known as gastrulation. Tissues such as, forexample, thyroid, thymus, pancreas, gut, and liver, will develop fromthe endoderm, via an intermediate stage. The intermediate stage in thisprocess is the formation of definitive endoderm. Definitive endodermcells express a number of markers, such as, HNF3 beta, GATA4, MIXL1,CXCR4 and SOX17.

Formation of the pancreas arises from the differentiation of definitiveendoderm into pancreatic endoderm. Cells of the pancreatic endodermexpress the pancreatic-duodenal homeobox gene, PDX1. In the absence ofPDX1, the pancreas fails to develop beyond the formation of ventral anddorsal buds. Thus, PDX1 expression marks a critical step in pancreaticorganogenesis. The mature pancreas contains, among other cell types,exocrine tissue and endocrine tissue. Exocrine and endocrine tissuesarise from the differentiation of pancreatic endoderm.

Cells bearing the features of islet cells have reportedly been derivedfrom embryonic cells of the mouse. For example, Lumelsky et al. (Science292:1389, 2001) report differentiation of mouse embryonic stem cells toinsulin-secreting structures similar to pancreatic islets. Soria et al.(Diabetes 49:157, 2000) report that insulin-secreting cells derived frommouse embryonic stem cells normalize glycemia in streptozotocin-induceddiabetic mice.

In one example, Hori et al. (PNAS 99: 16105, 2002) disclose thattreatment of mouse embryonic stem cells with inhibitors ofphosphoinositide 3-kinase (LY294002) produced cells that resembled βcells.

In another example, Blyszczuk et al. (PNAS 100:998, 2003) reports thegeneration of insulin-producing cells from mouse embryonic stem cellsconstitutively expressing Pax4.

Micallef et al. reports that retinoic acid can regulate the commitmentof embryonic stem cells to form PDX1 positive pancreatic endoderm.Retinoic acid is most effective at inducing Pdx1 expression when addedto cultures at day 4 of embryonic stem cell differentiation during aperiod corresponding to the end of gastrulation in the embryo (Diabetes54:301, 2005).

Miyazaki et al. reports a mouse embryonic stem cell line over-expressingPdx1. Their results show that exogenous Pdx1 expression clearly enhancedthe expression of insulin, somatostatin, glucokinase, neurogenin3, p48,Pax6, and Hnf6 genes in the resulting differentiated cells (Diabetes 53:1030, 2004).

Skoudy et al. reports that activin A (a member of the TGF-β superfamily)upregulates the expression of exocrine pancreatic genes (p48 andamylase) and endocrine genes (Pdx1, insulin, and glucagon) in mouseembryonic stem cells. The maximal effect was observed using 1 nM activinA. They also observed that the expression level of insulin and Pdx1 mRNAwas not affected by retinoic acid; however, 3 nM FGF7 treatment resultedin an increased level of the transcript for Pdx1 (Biochem. J. 379: 749,2004).

Shiraki et al. studied the effects of growth factors that specificallyenhance differentiation of embryonic stem cells into PDX1 positivecells. They observed that TGF-β2 reproducibly yielded a higherproportion of PDX1 positive cells (Genes Cells. 2005 June; 10(6):503-16.).

Gordon et al. demonstrated the induction of brachyury [positive]/HNF3beta [positive] endoderm cells from mouse embryonic stem cells in theabsence of serum and in the presence of activin along with an inhibitorof Wnt signaling (US 2006/0003446A1).

Gordon et al. (PNAS, Vol 103, page 16806, 2006) states “Wnt andTGF-beta/nodal/activin signaling simultaneously were required for thegeneration of the anterior primitive streak”.

However, the mouse model of embryonic stem cell development may notexactly mimic the developmental program in higher mammals, such as, forexample, humans.

Thomson et al. isolated embryonic stem cells from human blastocysts(Science 282:114, 1998). Concurrently, Gearhart and coworkers derivedhuman embryonic germ (hEG) cell lines from fetal gonadal tissue(Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Unlikemouse embryonic stem cells, which can be prevented from differentiatingsimply by culturing with Leukemia Inhibitory Factor (LIF), humanembryonic stem cells must be maintained under very special conditions(U.S. Pat. No. 6,200,806; WO 99/20741; WO 01/51616).

D'Amour et al. describes the production of enriched cultures of humanembryonic stem cell-derived definitive endoderm in the presence of ahigh concentration of activin and low serum (Nature Biotechnology 2005).Transplanting these cells under the kidney capsule of mice resulted indifferentiation into more mature cells with characteristics of someendodermal organs. Human embryonic stem cell-derived definitive endodermcells can be further differentiated into PDX1 positive cells afteraddition of FGF-10 (US 2005/0266554A1).

D'Amour et al. (Nature Biotechnology—24, 1392-1401 (2006)) states: “Wehave developed a differentiation process that converts human embryonicstem (hES) cells to endocrine cells capable of synthesizing thepancreatic hormones insulin, glucagon, somatostatin, pancreaticpolypeptide and ghrelin. This process mimics in vivo pancreaticorganogenesis by directing cells through stages resembling definitiveendoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursoren route to cells that express endocrine hormones”.

In another example, Fisk et al. reports a system for producingpancreatic islet cells from human embryonic stem cells(US2006/0040387A1). In this case, the differentiation pathway wasdivided into three stages. Human embryonic stem cells were firstdifferentiated to endoderm using a combination of sodium butyrate andactivin A. The cells were then cultured with TGF-β antagonists such asNoggin in combination with EGF or betacellulin to generate PDX1 positivecells. The terminal differentiation was induced by nicotinamide.

There still remains a significant need to develop in vitro methods togenerate a functional insulin expressing cell, that more closelyresemble a β cell. The present invention takes an alternative approachto improve the efficiency of differentiating human embryonic stem cellstoward insulin expressing cells, by generating a population of cellswherein greater than 85% of the cells in the population express markerscharacteristic of the definitive endoderm lineage.

SUMMARY

In one embodiment, the present invention provides a population of cells,wherein greater than 85% of the cells in the population express markerscharacteristic of the definitive endoderm lineage.

In embodiment, populations of pluripotent stem cells are differentiatedinto populations of cells expressing markers characteristic of thedefinitive endoderm lineage by culturing the pluripotent stem cells inmedium supplemented with BSA and a factor selected from the groupconsisting of insulin and IGF-1. In one embodiment, differentiation ofthe population of pluripotent stem cells toward a population of cellsexpressing markers characteristic of the definitive endoderm lineage isachieved by treating the pluripotent stem cells with activin A and a Wntligand.

In one embodiment, differentiation of the population of pluripotent stemcells toward a population of cells expressing markers characteristic ofthe definitive endoderm lineage is achieved by treating the pluripotentstem cells with GDF-8 and at least one other factor is selected from thegroup consisting of: an aniline-pyridinotriazine, a cyclicaniline-pyridinotriazine,N-{[1-(Phenylmethyl)azepan-4-yl]methyl}-2-pyridin-3-ylacetamide,4-{[4-(4-{[2-(Pyridin-2-ylamino)ethyl]amino}-1,3,5-triazin-2-yl)pyridin-2-yl]oxy}butan-1-ol,3-({3-[4-({2-[Methyl(pyridin-2-yl)amino]ethyl}amino)-1,3,5-triazin-2-yl]pyridin-2-yl}amino)propan-1-ol,N˜4˜-[2-(3-Fluorophenyl)ethyl]-N˜2˜-[3-(4-methylpiperazin-1-yl)propyl]pyrido[2,3-d]pyrimidine-2,4-diamine,1-Methyl-N-[(4-pyridin-3-yl-2-{[3-(trifluoromethyl)phenyl]amino}-1,3-thiazol-5-yl)methyl]piperidine-4-carboxamide,1,1-Dimethylethyl{2-[4-({5-[3-(3-hydroxypropyl)phenyl]-4H-1,2,4-triazol-3-yl}amino)phenyl]ethyl}carbamate,1,1-Dimethylethyl{[3-({5-[5-(3-hydroxypropyl)-2-(methyloxy)phenyl]-1,3-oxazol-2-yl}amino)phenyl]methyl}carbamate,1-({5-[6-({4-[(4-Methylpiperazin-1-yl)sulfonyl]phenyl}amino)pyrazin-2-yl]thiophen-2-yl}methyl)piperidin-4-ol,1-({4-[6-({4-[(4-Methylpiperazin-1-yl)sulfonyl]phenyl}amino)pyrazin-2-yl]thiophen-2-yl}methyl)piperidine-4-carboxamide,and2-{[4-(1-Methylethyl)phenyl]amino}-N-(2-thiophen-2-ylethyl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxamide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the real-time PCR analysis of the expression of the genesindicated in cells of the human embryonic stem cell line H1,differentiated according to the methods disclosed in Example 1.

FIG. 2 shows the FACS analysis of the expression of the proteinsindicated in cells of the human embryonic stem cell line H1,differentiated according to the methods disclosed in Example 1.

FIG. 3 shows the real-time PCR analysis of the expression of the genesindicated in cells of the human embryonic stem cell line H1,differentiated according to the methods disclosed in Example 2.

FIG. 4 shows the expression of SOX17 via immunofluorescence in cells ofthe human embryonic stem cell line H1, differentiated according to themethods disclosed in Example 2.

FIG. 5 shows the FACS analysis of the expression of the proteinsindicated in cells of the human embryonic stem cell line H1,differentiated according to the methods disclosed in Example 2.

FIG. 6 shows the real-time PCR analysis of the expression of the genesindicated in cells of the human embryonic stem cell line H1,differentiated according to the methods disclosed in Example 3.

FIG. 7 shows the expression of SOX17 via immunofluorescence in cells ofthe human embryonic stem cell line H1, differentiated according to themethods disclosed in Example 3.

FIG. 8 shows the expression of SOX17 via immunofluorescence in cells ofthe human embryonic stem cell line H1, differentiated according to themethods disclosed in Example 3.

FIG. 9 shows the real-time PCR analysis of the expression of the genesindicated in cells of the human embryonic stem cell line H1,differentiated according to the methods disclosed in Example 5.

DETAILED DESCRIPTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the following subsectionsthat describe or illustrate certain features, embodiments orapplications of the present invention.

DEFINITIONS

Stem cells are undifferentiated cells defined by their ability at thesingle cell level to both self-renew and differentiate to produceprogeny cells, including self-renewing progenitors, non-renewingprogenitors, and terminally differentiated cells. Stem cells are alsocharacterized by their ability to differentiate in vitro into functionalcells of various cell lineages from multiple germ layers (endoderm,mesoderm and ectoderm), as well as to give rise to tissues of multiplegerm layers following transplantation and to contribute substantially tomost, if not all, tissues following injection into blastocysts.

Stem cells are classified by their developmental potential as: (1)totipotent, meaning able to give rise to all embryonic andextraembryonic cell types; (2) pluripotent, meaning able to give rise toall embryonic cell types; (3) multipotent, meaning able to give rise toa subset of cell lineages but all within a particular tissue, organ, orphysiological system (for example, hematopoietic stem cells (HSC) canproduce progeny that include HSC (self-renewal), blood cell restrictedoligopotent progenitors, and all cell types and elements (e.g.,platelets) that are normal components of the blood); (4) oligopotent,meaning able to give rise to a more restricted subset of cell lineagesthan multipotent stem cells; and (5) unipotent, meaning able to giverise to a single cell lineage (e.g., spermatogenic stem cells).

Differentiation is the process by which an unspecialized (“uncommitted”)or less specialized cell acquires the features of a specialized cellsuch as, for example, a nerve cell or a muscle cell. A differentiated ordifferentiation-induced cell is one that has taken on a more specialized(“committed”) position within the lineage of a cell. The term“committed”, when applied to the process of differentiation, refers to acell that has proceeded in the differentiation pathway to a point where,under normal circumstances, it will continue to differentiate into aspecific cell type or subset of cell types, and cannot, under normalcircumstances, differentiate into a different cell type or revert to aless differentiated cell type. De-differentiation refers to the processby which a cell reverts to a less specialized (or committed) positionwithin the lineage of a cell. As used herein, the lineage of a celldefines the heredity of the cell, i.e., which cells it came from andwhat cells it can give rise to. The lineage of a cell places the cellwithin a hereditary scheme of development and differentiation. Alineage-specific marker refers to a characteristic specificallyassociated with the phenotype of cells of a lineage of interest and canbe used to assess the differentiation of an uncommitted cell to thelineage of interest.

“Cells expressing markers characteristic of the definitive endodermlineage”, or “Stage 1 cells”, or “Stage 1”, as used herein, refers tocells expressing at least one of the following markers: SOX17, GATA4,HNF3 beta, GSC, CER1, Nodal, FGF8, Brachyury, Mix-like homeobox protein,FGF4 CD48, eomesodermin (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99,or OTX2. Cells expressing markers characteristic of the definitiveendoderm lineage include primitive streak precursor cells, primitivestreak cells, mesendoderm cells and definitive endoderm cells.

“Cells expressing markers characteristic of the pancreatic endodermlineage”, as used herein, refers to cells expressing at least one of thefollowing markers: PDX1, NKX6.1, HNF1 beta, PTF1 alpha, HNF6, HNF4alpha, SOX9, HB9 or PROX1. Cells expressing markers characteristic ofthe pancreatic endoderm lineage include pancreatic endoderm cells,primitive gut tube cells, and posterior foregut cells.

“Definitive endoderm”, as used herein, refers to cells which bear thecharacteristics of cells arising from the epiblast during gastrulationand which form the gastrointestinal tract and its derivatives.Definitive endoderm cells express the following markers: HNF3 beta,GATA4, SOX17, Cerberus, OTX2, goosecoid, C-Kit, CD99, and MIXL1.

“Markers”, as used herein, are nucleic acid or polypeptide moleculesthat are differentially expressed in a cell of interest. In thiscontext, differential expression means an increased level for a positivemarker and a decreased level for a negative marker. The detectable levelof the marker nucleic acid or polypeptide is sufficiently higher orlower in the cells of interest compared to other cells, such that thecell of interest can be identified and distinguished from other cellsusing any of a variety of methods known in the art.

“Pancreatic endocrine cell”, or “Pancreatic hormone expressing cell”, or“Cells expressing markers characteristic of the pancreatic endocrinelineage” as used herein, refers to a cell capable of expressing at leastone of the following hormones: insulin, glucagon, somatostatin, andpancreatic polypeptide.

Isolation, Expansion and Culture of Pluripotent Stem CellsCharacterization of Pluripotent Stem Cells

Pluripotent stem cells may express one or more of the stage-specificembryonic antigens (SSEA) 3 and 4, and markers detectable usingantibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science282:1145, 1998). Differentiation of pluripotent stem cells in vitroresults in the loss of SSEA-4, Tra 1-60, and Tra 1-81 expression (ifpresent) and increased expression of SSEA-1. Undifferentiatedpluripotent stem cells typically have alkaline phosphatase activity,which can be detected by fixing the cells with 4% paraformaldehyde, andthen developing with Vector Red as a substrate, as described by themanufacturer (Vector Laboratories, Burlingame Calif.). Undifferentiatedpluripotent stem cells also typically express OCT4 and TERT, as detectedby RT-PCR.

Another desirable phenotype of propagated pluripotent stem cells is apotential to differentiate into cells of all three germinal layers:endoderm, mesoderm, and ectoderm tissues. Pluripotency of pluripotentstem cells can be confirmed, for example, by injecting cells into severecombined immunodeficient (SCID) mice, fixing the teratomas that formusing 4% paraformaldehyde, and then examining them histologically forevidence of cell types from the three germ layers. Alternatively,pluripotency may be determined by the creation of embryoid bodies andassessing the embryoid bodies for the presence of markers associatedwith the three germinal layers.

Propagated pluripotent stem cell lines may be karyotyped using astandard G-banding technique and compared to published karyotypes of thecorresponding primate species. It is desirable to obtain cells that havea “normal karyotype,” which means that the cells are euploid, whereinall human chromosomes are present and not noticeably altered.

Sources of Pluripotent Stem Cells

The types of pluripotent stem cells that may be used include establishedlines of pluripotent cells derived from tissue formed after gestation,including pre-embryonic tissue (such as, for example, a blastocyst),embryonic tissue, or fetal tissue taken any time during gestation,typically but not necessarily before approximately 10 to 12 weeksgestation. Non-limiting examples are established lines of humanembryonic stem cells or human embryonic germ cells, such as, for examplethe human embryonic stem cell lines H1, H7, and H9 (WiCell). Alsocontemplated is use of the compositions of this disclosure during theinitial establishment or stabilization of such cells, in which case thesource cells would be primary pluripotent cells taken directly from thesource tissues. Also suitable are cells taken from a pluripotent stemcell population already cultured in the absence of feeder cells. Alsosuitable are mutant human embryonic stem cell lines, such as, forexample, BG01v (BresaGen, Athens, Ga.).

In one embodiment, human embryonic stem cells are prepared as describedby Thomson et al. (U.S. Pat. No. 5,843,780; Science 282:1145, 1998;Curr. Top. Dev. Biol. 38:133 ff., 1998; Proc. Natl. Acad. Sci. U.S.A.92:7844, 1995).

Culture of Pluripotent Stem Cells

In one embodiment, pluripotent stem cells are cultured on a layer offeeder cells that support the pluripotent stem cells in various ways.Alternatively, pluripotent stem cells are cultured in a culture systemthat is essentially free of feeder cells, but nonetheless supportsproliferation of pluripotent stem cells without undergoing substantialdifferentiation. The growth of pluripotent stem cells in feeder-freeculture without differentiation is supported using a medium conditionedby culturing previously with another cell type. Alternatively, thegrowth of pluripotent stem cells in feeder-free culture withoutdifferentiation is supported using a chemically defined medium.

In one embodiment, pluripotent stem cells may be cultured on a mouseembryonic fibroblast feeder cell layer according to the methodsdisclosed in Reubinoff et al (Nature Biotechnology 18: 399-404 (2000)).Alternatively, pluripotent stem cells may be cultured on a mouseembryonic fibroblast feeder cell layer according to the methodsdisclosed in Thompson et al (Science 6 Nov. 1998: Vol. 282. no. 5391,pp. 1145-1147). Alternatively, pluripotent stem cells may be cultured onany one of the feeder cell layers disclosed in Richards et al, (StemCells 21: 546-556, 2003).

In one embodiment, pluripotent stem cells may be cultured on a humanfeeder cell layer according to the methods disclosed in Wang et al (StemCells 23: 1221-1227, 2005). In an alternate embodiment, pluripotent stemcells may be cultured on the human feeder cell layer disclosed inStojkovic et al (Stem Cells 2005 23: 306-314, 2005). Alternatively,pluripotent stem cells may be cultured on the human feeder cell layerdisclosed in Miyamoto et al (Stem Cells 22: 433-440, 2004).Alternatively, pluripotent stem cells may be cultured on the humanfeeder cell layer disclosed in Amit et al (Biol. Reprod 68: 2150-2156,2003). Alternatively, pluripotent stem cells may be cultured on thehuman feeder cell layer disclosed in Inzunza et al (Stem Cells 23:544-549, 2005).

In one embodiment, pluripotent stem cells may be cultured in culturemedia derived according to the methods disclosed in US20020072117.Alternatively, pluripotent stem cells may be cultured in culture mediaderived according to the methods disclosed in U.S. Pat. No. 6,642,048.Alternatively, pluripotent stem cells may be cultured in culture mediaderived according to the methods disclosed in WO2005014799.Alternatively, pluripotent stem cells may be cultured in culture mediaderived according to the methods disclosed in Xu et al (Stem Cells 22:972-980, 2004). Alternatively, pluripotent stem cells may be cultured inculture media derived according to the methods disclosed inUS20070010011. Alternatively, pluripotent stem cells may be cultured inculture media derived according to the methods disclosed inUS20050233446. Alternatively, pluripotent stem cells may be cultured inculture media derived according to the methods disclosed in U.S. Pat.No. 6,800,480. Alternatively, pluripotent stem cells may be cultured inculture media derived according to the methods disclosed inWO2005065354.

In one embodiment, pluripotent stem cells may be cultured according tothe methods disclosed in Cheon et al (BioReprodDOI:10.1095/biolreprod.105.046870, Oct. 19, 2005). Alternatively,pluripotent stem cells may be cultured according to the methodsdisclosed in Levenstein et al (Stem Cells 24: 568-574, 2006).Alternatively, pluripotent stem cells may be cultured according to themethods disclosed in US20050148070. Alternatively, pluripotent stemcells may be cultured according to the methods disclosed inUS20050244962. Alternatively, pluripotent stem cells may be culturedaccording to the methods disclosed in WO2005086845.

The pluripotent stem cells may be plated onto a suitable culturesubstrate. In one embodiment, the suitable culture substrate is anextracellular matrix component, such as, for example, those derived frombasement membrane or that may form part of adhesion moleculereceptor-ligand couplings. In one embodiment, the suitable culturesubstrate is MATRIGEL® (Becton Dickenson). MATRIGEL® is a solublepreparation from Engelbreth-Holm Swarm tumor cells that gels at roomtemperature to form a reconstituted basement membrane.

Other extracellular matrix components and component mixtures aresuitable as an alternative. Depending on the cell type beingproliferated, this may include laminin, fibronectin, proteoglycan,entactin, heparan sulfate, and the like, alone or in variouscombinations.

The pluripotent stem cells may be plated onto the substrate in asuitable distribution and in the presence of a medium that promotes cellsurvival, propagation, and retention of the desirable characteristics.All these characteristics benefit from careful attention to the seedingdistribution and can readily be determined by one of skill in the art.

Suitable culture media may be made from the following components, suchas, for example, Dulbecco's modified Eagle's medium (DMEM), Gibco#11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco#10829-018; Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco#15039-027; non-essential amino acid solution, Gibco 11140-050;β-mercaptoethanol, Sigma #M7522; human recombinant basic fibroblastgrowth factor (bFGF), Gibco #13256-029.

Formation of Cells Expressing Markers Characteristic of the DefinitiveEndoderm Lineage from Pluripotent Stem Cells

The present invention provides methods for the formation of populationsof cells expressing markers characteristic of the definitive endodermlineage from populations of pluripotent stem cells. In one embodiment,the present invention provides methods to further differentiate thecells expressing markers characteristic of the definitive endodermlineage into cells expressing markers of the pancreatic endocrinelineage. In one embodiment, this is achieved utilizing a step-wisedifferentiation protocol, wherein populations of pluripotent stem cellsare first differentiated into populations of cells expressing markerscharacteristic of the definitive endoderm lineage. Next, the populationsof cells expressing markers characteristic of the definitive endodermlineage are then differentiated into populations of cells expressingmarkers characteristic of the pancreatic endoderm lineage. Next, thepopulations of cells expressing markers characteristic of the pancreaticendoderm lineage are then differentiated into populations of cellsexpressing markers characteristic of the pancreatic endocrine lineage.

The present invention provides a population of cells wherein greaterthan 85% of the cells express markers characteristic of the definitiveendoderm lineage. The population of cells may be further treated to forma population of cells expressing markers characteristic of thepancreatic endoderm lineage. The population of cells expressing markerscharacteristic of the pancreatic endoderm lineage may be further treatedto form a population of cells expressing markers characteristic of thepancreatic endocrine lineage.

The efficiency of differentiation may be determined by exposing atreated cell population to an agent (such as an antibody) thatspecifically recognizes a protein marker expressed by cells expressingmarkers characteristic of the desired cell type.

Methods for assessing expression of protein and nucleic acid markers incultured or isolated cells are standard in the art. These includequantitative reverse transcriptase polymerase chain reaction (RT-PCR),Northern blots, in situ hybridization (see, e.g., Current Protocols inMolecular Biology (Ausubel et al., eds. 2001 supplement)), andimmunoassays such as immunohistochemical analysis of sectioned material,Western blotting, and for markers that are accessible in intact cells,flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, UsingAntibodies: A Laboratory Manual, New York: Cold Spring Harbor LaboratoryPress (1998)).

Characteristics of pluripotent stem cells are well known to thoseskilled in the art, and additional characteristics of pluripotent stemcells continue to be identified. Pluripotent stem cell markers include,for example, the expression of one or more of the following: ABCG2,cripto, FOXD3, CONNEXIN43, CONNEXIN45, OCT4, SOX2, NANOG, hTERT, UTF1,ZFP42, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81.

After treating pluripotent stem cells with the methods of the presentinvention, the differentiated cells may be purified by exposing atreated cell population to an agent (such as an antibody) thatspecifically recognizes a protein marker, such as CXCR4, expressed bycells expressing markers characteristic of the definitive endodermlineage.

Pluripotent stem cells suitable for use in the present inventioninclude, for example, the human embryonic stem cell line H9 (NIH code:WA09), the human embryonic stem cell line H1 (NIH code: WA01), the humanembryonic stem cell line H7 (NIH code: WA07), and the human embryonicstem cell line SA002 (Cellartis, Sweden). Also suitable for use in thepresent invention are cells that express at least one of the followingmarkers characteristic of pluripotent cells: ABCG2, cripto, CD9, FOXD3,CONNEXIN43, CONNEXIN45, OCT4, SOX2, NANOG, hTERT, UTF1, ZFP42, SSEA-3,SSEA-4, Tra 1-60, and Tra 1-81.

Markers characteristic of the definitive endoderm lineage are selectedfrom the group consisting of SOX17, GATA4, HNF3 beta, GSC, CER1, Nodal,FGF8, Brachyury, Mix-like homeobox protein, FGF4, CD48, eomesodermin(EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99, and OTX2. Suitable foruse in the present invention is a cell that expresses at least one ofthe markers characteristic of the definitive endoderm lineage. In oneaspect of the present invention, a cell expressing markerscharacteristic of the definitive endoderm lineage is a primitive streakprecursor cell. In an alternate aspect, a cell expressing markerscharacteristic of the definitive endoderm lineage is a mesendoderm cell.In an alternate aspect, a cell expressing markers characteristic of thedefinitive endoderm lineage is a definitive endoderm cell.

Markers characteristic of the pancreatic endoderm lineage are selectedfrom the group consisting of PDX1, NKX6.1, HNF1 beta, PTF1 alpha, HNF6,HNF4 alpha, SOX9, HB9 and PROX1. Suitable for use in the presentinvention is a cell that expresses at least one of the markerscharacteristic of the pancreatic endoderm lineage. In one aspect of thepresent invention, a cell expressing markers characteristic of thepancreatic endoderm lineage is a pancreatic endoderm cell.

Markers characteristic of the pancreatic endocrine lineage are selectedfrom the group consisting of NGN3, NEUROD, ISL1, PDX1, NKX6.1, PAX4,NGN3, and PTF1 alpha. In one embodiment, a pancreatic endocrine cell iscapable of expressing at least one of the following hormones: insulin,glucagon, somatostatin, and pancreatic polypeptide. Suitable for use inthe present invention is a cell that expresses at least one of themarkers characteristic of the pancreatic endocrine lineage. In oneaspect of the present invention, a cell expressing markerscharacteristic of the pancreatic endocrine lineage is a pancreaticendocrine cell. The pancreatic endocrine cell may be a pancreatichormone-expressing cell. Alternatively, the pancreatic endocrine cellmay be a pancreatic hormone-secreting cell.

In one aspect of the present invention, the pancreatic endocrine cell isa cell expressing markers characteristic of the β cell lineage. A cellexpressing markers characteristic of the β cell lineage expresses PDX1and at least one of the following transcription factors: NGN3, NKX2.2,NKX6.1, NEUROD, ISL1, HNF3 beta, MAFA, PAX4, and PAX6. In one aspect ofthe present invention, a cell expressing markers characteristic of the βcell lineage is a β cell.

Formation of Cells Expressing Markers Characteristic of the DefinitiveEndoderm Lineage from Pluripotent Stem Cells

In one aspect of the present invention, populations of pluripotent stemcells may be differentiated into populations of cells expressing markerscharacteristic of the definitive endoderm lineage by culturing thepluripotent stem cells in medium lacking serum and supplemented with BSAand a factor selected from the group consisting of insulin and IGF-1. Inone embodiment, differentiation of the population of pluripotent stemcells toward a population of cells expressing markers characteristic ofthe definitive endoderm lineage is achieved by treating the pluripotentstem cells with activin A and a Wnt ligand.

In an alternate embodiment, differentiation of the population ofpluripotent stem cells toward a population of cells expressing markerscharacteristic of the definitive endoderm lineage is achieved bytreating the pluripotent stem cells with GDF-8 and at least one otherfactor is selected from the group consisting of: ananiline-pyridinotriazine, a cyclic aniline-pyridinotriazine,N-{[1-(Phenylmethyl)azepan-4-yl]methyl}-2-pyridin-3-ylacetamide,4-{[4-(4-{[2-(Pyridin-2-ylamino)ethyl]amino}-1,3,5-triazin-2-yl)pyridin-2-yl]oxy}butan-1-ol,3-({3-[4-({2-[Methyl(pyridin-2-yl)amino]ethyl}amino)-1,3,5-triazin-2-yl]pyridin-2-yl}amino)propan-1-ol,N˜4˜-[2-(3-Fluorophenyl)ethyl]-N˜2˜-[3-(4-methylpiperazin-1-yl)propyl]pyrido[2,3-d]pyrimidine-2,4-diamine,1-Methyl-N-[(4-pyridin-3-yl-2-{[3-(trifluoromethyl)phenyl]amino}-1,3-thiazol-5-yl)methyl]piperidine-4-carboxamide,1,1-Dimethylethyl{2-[4-({5-[3-(3-hydroxypropyl)phenyl]-4H-1,2,4-triazol-3-yl}amino)phenyl]ethyl}carbamate,1,1-Dimethylethyl{[3-({5-[5-(3-hydroxypropyl)-2-(methyloxy)phenyl]-1,3-oxazol-2-yl}amino)phenyl]methyl}carbamate,1-({5-[6-({4-[4-Methylpiperazin-1-yl)sulfonyl]phenyl}amino)pyrazin-2-yl]thiophen-2-yl}methyl)piperidin-4-ol,1-({4-[6-({4-[4-Methylpiperazin-1-yl)sulfonyl]phenyl}amino)pyrazin-2-yl]thiophen-2-yl}methyl)piperidine-4-carboxamide,and2-{[4-(1-Methylethyl)phenyl]amino}-N-(2-thiophen-2-ylethyl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxamide.Examples of the factors suitable for use may be found in U.S. patentapplication Ser. No. 12/494,789. In one embodiment, the at least oneother factor is14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜0.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one.

The population of pluripotent stem cells may be cultured in the mediumlacking serum and supplemented with BSA and a factor selected from thegroup consisting of insulin and IGF-1 for about one day to about sevendays. Alternatively, the population of pluripotent stem cells may becultured in the medium lacking serum and supplemented with BSA and afactor selected from the group consisting of insulin and IGF-1 for aboutone day to about six days. Alternatively, the population of pluripotentstem cells may be cultured in the medium lacking serum and supplementedwith BSA and a factor selected from the group consisting of insulin andIGF-1 for about one day to about five days. Alternatively, thepopulation of pluripotent stem cells may be cultured in the mediumlacking serum and supplemented with BSA and a factor selected from thegroup consisting of insulin and IGF-1 for about one day to about fourdays. Alternatively, the population of pluripotent stem cells may becultured in the medium lacking serum and supplemented with BSA and afactor selected from the group consisting of insulin and IGF-1 for aboutfour days.

In one embodiment, the GDF-8 is used at a concentration from about 5ng/ml to about 500 ng/ml. In an alternate embodiment, the GDF-8 is usedat a concentration from about 5 ng/ml to about 50 ng/ml. In an alternateembodiment, the GDF-8 is used at a concentration from about 5 ng/ml toabout 25 ng/ml. In an alternate embodiment, the GDF-8 is used at aconcentration of about 25 ng/ml.

Activin-A may be used at a concentration from about 1 pg/ml to about 100μg/ml. In an alternate embodiment, the concentration may be about 1pg/ml to about 1 μg/ml. In another alternate embodiment, theconcentration may be about 1 pg/ml to about 100 ng/ml. In anotheralternate embodiment, the concentration may be about 50 ng/ml to about100 ng/ml. In another alternate embodiment, the concentration may beabout 100 ng/ml.

The Wnt ligand may be selected from the group consisting of Wnt-1,Wnt-3a, Wnt-5a and Wnt-7a. In one embodiment, the Wnt ligand is Wnt-1.In an alternate embodiment, the Wnt ligand is Wnt-3a.

The Wnt ligand may be used at a concentration of about 1 ng/ml to about1000 ng/ml. In an alternate embodiment, the Wnt ligand may be used at aconcentration of about 10 ng/ml to about 100 ng/ml. In one embodiment,the concentration of the Wnt ligand is about 20 ng/ml.

In one embodiment, insulin is used at a concentration from about 1 ng/mlto about 100 ng/ml.

In one embodiment, IGF-1 is used at a concentration from about 1 ng/mlto about 200 ng/ml.

Formation of Cells Expressing Markers Characteristic of the PancreaticEndoderm Lineage

In one embodiment, populations of cells expressing markerscharacteristic of the definitive endoderm lineage formed by the methodsof the present invention are further differentiated into populations ofcells expressing markers characteristic of the pancreatic endodermlineage by any method in the art.

For example, populations of cells expressing markers characteristic ofthe definitive endoderm lineage obtained according to the methods of thepresent invention may be further differentiated into populations ofcells expressing markers characteristic of the pancreatic endodermlineage by treating the population of cells expressing markerscharacteristic of the definitive endoderm lineage according to themethods disclosed in D'Amour et al, Nature Biotechnology 24, 1392-1401(2006).

For example, populations of cells expressing markers characteristic ofthe definitive endoderm lineage obtained according to the methods of thepresent invention may be further differentiated into populations ofcells expressing markers characteristic of the pancreatic endodermlineage by treating the population of cells expressing markerscharacteristic of the definitive endoderm lineage according to themethods disclosed in U.S. patent application Ser. No. 11/736,908.

Formation of Cells Expressing Markers Characteristic of the PancreaticEndocrine Lineage

In one embodiment, populations of cells expressing markerscharacteristic of the pancreatic endoderm lineage are furtherdifferentiated into populations of cells expressing markerscharacteristic of the pancreatic endocrine lineage by any method in theart.

For example, populations of cells expressing markers characteristic ofthe pancreatic endoderm lineage may be further differentiated intopopulations of cells expressing markers characteristic of the pancreaticendocrine lineage, by treating the population of cells expressingmarkers characteristic of the pancreatic endoderm lineage according tothe methods disclosed in D′ Amour et al, Nature Biotechnology, 2006.

For example, populations of cells expressing markers characteristic ofthe pancreatic endoderm lineage may be further differentiated intopopulations of cells expressing markers characteristic of the pancreaticendocrine lineage, by treating the population of cells expressingmarkers characteristic of the pancreatic endoderm lineage according tothe methods disclosed in D′ Amour et al, Nature Biotechnology, 2006.

For example, populations of cells expressing markers characteristic ofthe pancreatic endoderm lineage may be further differentiated intopopulations of cells expressing markers characteristic of the pancreaticendocrine lineage, by treating the population of cells expressingmarkers characteristic of the pancreatic endoderm lineage according tothe methods disclosed in U.S. patent application Ser. No. 11/736,908.

For example, populations of cells expressing markers characteristic ofthe pancreatic endoderm lineage may be further differentiated intopopulations of cells expressing markers characteristic of the pancreaticendocrine lineage, by treating the population of cells expressingmarkers characteristic of the pancreatic endoderm lineage according tothe methods disclosed in U.S. patent application Ser. No. 11/779,311.

For example, populations of cells expressing markers characteristic ofthe pancreatic endoderm lineage may be further differentiated intopopulations of cells expressing markers characteristic of the pancreaticendocrine lineage, by treating the population of cells expressingmarkers characteristic of the pancreatic endoderm lineage according tothe methods disclosed in U.S. patent application Ser. No. 60/953,178.

For example, populations of cells expressing markers characteristic ofthe pancreatic endoderm lineage may be further differentiated intopopulations of cells expressing markers characteristic of the pancreaticendocrine lineage, by treating the population of cells expressingmarkers characteristic of the pancreatic endoderm lineage according tothe methods disclosed in U.S. patent application Ser. No. 60/990,529.

The present invention is further illustrated, but not limited by, thefollowing examples.

EXAMPLES Example 1 The Role of Insulin in the Differentiation of HumanPluripotent Stem Cells to Cells Expressing Markers Characteristic of theDefinitive Endoderm Lineage: Cluster Seeding

Previous studies have shown that a high concentration of FBS isdetrimental to the formation of definitive endoderm (DE) from embryonicstem cells. See, for example, D'Amour et al., Nature Biotechnology,2005, where the induction of definitive endoderm from human embryonicstem cells was significantly increased when the FBS concentration wasreduced from 10% FBS to 0.5-2% FBS. Similar observations were reported,wherein addition of 25 ng/ml of IGF or 200 ng/ml of insulin to 2% FBS toES cells cultured in MEF-CM (mouse embryonic fibroblast conditionedmedia) decreased the expression of SOX17 by approximately 70% followingtreatment with activin A. See McLean et al, Stem Cells 25:29-38, 2007.

The inhibitory effect observed was likely due to the presence of insulinor IGF in the FBS, triggering the Phosphatidylinositol 3-Kinase pathway.See McLean et al, Stem Cells 25:29-38, 2007. Blockade of the PI-3 kinasesignaling pathway increased percentage of Sox17 positive cells in humanES cells cultured in MEF-CM (mouse embryonic fibroblast conditionedmedia). See McLean et al, Stem Cells 25:29-38, 2007.

These data suggest that it would be expected that addition of as littleas 25 ng/ml of IGF or 200 ng/ml of insulin to media containing activin Aand low concentration of FBS (0.5-2% FBS) would block the formation ofdefinitive endoderm. Typical concentration of IGF and insulin in FBS isapproximately 70 ng/ml (J. Clin. Invest. 76:4, 1985) and approximately60 ng/ml (In Vitro Cell Dev Biol. 32:8-12, 1996), respectively. Thistranslates to approximately 1.4 ng/ml of IGF and approximately 1.2 ng/mlof insulin in 2% FBS.

Cells of the human embryonic stem cells line H1 (p40-p52) were culturedon MATRIGEL® (1:30 dilution) (BD Biosciences; Cat #356231)-coated dishesin MEF-CM (mouse embryonic fibroblast conditioned media) supplementedwith 16 ng/ml of FGF2 (Catalog#100-18B, PeproTech, NJ), anddifferentiated into cells expressing markers characteristic of thedefinitive endoderm lineage as follows:

-   -   a. RPMI medium supplemented with 2% fatty acid-free BSA        (Catalog#68700, Proliant, IA), and 100 ng/ml activin A (R&D        Systems, MN) plus 20 ng/ml WNT-3a (Catalog#1324-WN-002, R&D        Systems, MN) for one day, then    -   b. RPMI medium supplemented with 2% BSA and 100 ng/ml activin A        for an additional three days.

In some of the cultures, the cells were treated with the followingdilution of ITS-X (Catalogue#51500-056, Invitrogen, CA): 0, 1:10⁶,1:5×10⁵, 1:10⁵, 1:10⁴. ITS-X is a serum replacement supplementedcomprised of 1 mg/ml of Insulin, 0.55 mg/ml of Transferrin, 0.00067mg/ml of Sodium Selenite, and 0.2 mg/ml of Ethanolamine The range ofdilutions of ITS-X correspond to 0, 1 ng/ml, 2 ng/ml, 10 ng/ml, and 100ng/ml of insulin. As a control, 0.2% FBS (Catalogue#SH30070.03, Hyclone,UT) was used for day 1 of differentiation, 0.5% FBS at day 2 and 2% FBSwas used for days 3-4. The FBS treated cultures were not supplementedwith ITS-X.

At day 4, samples were collected for FACS and gene expression analysisusing real-time PCR. Surprisingly, as shown in FIG. 1, addition of 1-100ng/ml of insulin to the medium used to differentiate the cells, did notsignificantly affect the expression of markers associated withdefinitive endoderm (FOXA2, SOX17, and CXCR4), markers associated withmesenchyme (T, also known as Brach), or extraembryonic markers (SOX7,AFP). Furthermore, cultures treated with medium supplemented with 2% BSAshowed significantly higher expression of markers associated withdefinitive endoderm, than cultures treated with medium supplemented with0.5-2% FBS.

These observations were further supported by the expression of CXCR4 andCD9, as determined by FACS, for the various treatments. See FIG. 2. Thecell surface receptor CXCR4 has been previously shown to be a marker ofdefinitive endoderm. CD9 is a marker for undifferentiated ES cells.Consequently, an increase in the expression of CXCR4, and a decrease inthe expression of CD9 in a population of cells is indicative for theformation of definitive endoderm. As summarized in Table I, nosignificant change in the expression of CXCR4, or CD9 was observed incells treated with medium supplemented with BSA, at any concentration ofinsulin tested. These data suggest that insulin is not inhibitory at theconcentrations tested in the culture medium employed in these studies.

TABLE I Treatment % CXCR4 + CD9− % CXCR4 − CD9+ % CXCR4 − CD9− FBS 56 279 BSA 69 13 13 BSA + 1 70 13 10 ng/ml insulin BSA + 5 67 15 12 ng/mlinsulin BSA + 10 69 13 13 ng/ml insulin BSA + 100 73 12 9 ng/ml insulin

Example 2 The Role of Insulin in the Differentiation of HumanPluripotent Stem Cells to Cells Expressing Markers Characteristic of theDefinitive Endoderm Lineage: Single Cell Seeding

Cells of the human embryonic stem cells line H1 (p40-p52) were seeded assingle cells at a density of 100000 cells/cm² on MATRIGEL® (1:30dilution) (BD Biosciences; Cat #356231)-coated dishes in MEF-CM (mouseembryonic fibroblast conditioned media) supplemented with 16 ng/ml ofFGF2 (Catalog#100-18B, PeproTech, NJ) and 10 μM of Y27632 (Rockinhibitor, Catalogue#Y0503, Sigma, Mo.). 72 hrs post seeding, cultureswere differentiated into definitive endoderm (DE) as follows:

-   -   a. MCDB-131 (Catalogue#10372-019, Invitrogen, CA) medium        supplemented with 2% fatty acid-free BSA (Catalog#68700,        Proliant, IA), 0.0025 g/ml sodium bicarbonate (Catalogue #S3187,        Sigma, Mo.), 1× GlutaMax™ (Catalogue #35050-079, Invitrogen, Ca)        and 100 ng/ml activin A (R&D Systems, MN) plus 20 ng/ml WNT-3a        (Catalog#1324-WN-002, R&D Systems, MN) for one day, then    -   b. MCDB-131 medium supplemented with 2% BSA, sodium bicarbonate,        Glutamax, and 100 ng/ml activin A for an additional three days.

In some of the cultures, the cells were treated with the followingconcentrations of insulin (Catalogue#19278, Sigma, Mo.): 0, 1, 10, 100,1000, or 10000 ng/ml. At day 4, samples were collected for FACS and geneexpression analysis using real-time PCR.

Addition of 1-100 ng/ml insulin to the medium used to differentiate thecells, did not significantly affect the expression of markers associatedwith definitive endoderm (FOXA2, SOX17, CER1, and CXCR4). Similarly, theexpression of embryonic markers (NANOG), or extraembryonic markers(SOX7, AFP) were not affected. See FIG. 3. Addition of 1-10 μg/ml ofinsulin, however, did increase expression of NANOG. These data werefurther supported by immuno fluorescence (IF) staining for thedefinitive endoderm marker SOX17 (Catalogue #AF1924, R & D systems, MN)(FIG. 4).

FIG. 5 depicts the CXCR4 and CD9 expression profile of the varioustreatments as measured by FACS analysis. As summarized in Table II, onlyat super physiological concentrations of insulin (1-10 μg/ml) there wasa decrease in the percentage of CXCR4+CD9− cells and an increase inexpression of CXCR4−CD9+ fraction. These data suggest that in theconditions in this study, only superphysiological concentrations ofinsulin inhibit the formation of definitive endoderm.

TABLE II Treatment % CXCR4 + CD9− % CXCR4 − CD9+ % CXCR4 − CD9− BSA 961.3 0.9 BSA + 1 96 1.5 0.7 ng/ml insulin BSA + 10 95 1.4 0.7 ng/mlinsulin BSA + 100 90 4.1 2 ng/ml insulin BSA + 1 90 3.6 2.2 μg/mlinsulin BSA + 10 84 6.6 4.7 μg/ml insulin

Example 3 The Role of IGF in the Differentiation of Human PluripotentStem Cells to Cells Expressing Markers Characteristic of the DefinitiveEndoderm Lineage: Single Cell Seeding

Cells of the human embryonic stem cells line H1 (p40-p52) were seeded assingle cells at a density of 100000 cells/cm² on MATRIGEL® (1:30dilution) (BD Biosciences; Cat #356231)-coated dishes in MEF-CM (mouseembryonic fibroblast conditioned media) supplemented with 16 ng/ml ofFGF2 (Catalog#100-18B, PeproTech, NJ) and 10 μM of Y27632 (Rockinhibitor, Catalogue#Y0503, Sigma, Mo.). 72 hrs post seeding, cultureswere differentiated into definitive endoderm (DE) as follows:

-   -   a. MCDB-131 (Catalogue#10372-019, Invitrogen, CA) medium        supplemented with 2% fatty acid-free BSA (Catalog#68700,        Proliant, IA), 0.0025 g/ml sodium bicarbonate (Catalogue #S3187,        Sigma, Mo.), 1× GlutaMax™ (Catalogue #35050-079, Invitrogen, Ca)        and 100 ng/ml GDF8 (Catalogue#120-00, PeproTech, NJ) plus 2.5 μM        of the GSK3B inhibitor        14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo        [19.3.1.1˜2,6˜0.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one        for one day, then    -   b. MCDB-131 medium supplemented with 2% BSA, sodium bicarbonate,        Glutamax, and 100 ng/ml GDF-8 for an additional three days.

In some of the cultures, the cells were treated with the followingconcentrations of IGF (Catalogue#AF100, PeproTech, NJ): 0, 1, 10, 50, or200 ng/ml. As a control, instead of BSA, 0.2% FBS (Catalogue#SH30070.03,Hyclone, UT) was used for day 1 of differentiation, and 2% FBS was usedfor days 2-4. Some of the FBS treated cultures were also treated withvarious concentrations of IGF.

At day 4, samples were collected for FACS and gene expression analysisusing real-time PCR.

Surprisingly, addition of 1-200 ng/ml of IGF to BSA treated cultures didnot significantly affect the expression of expression of markersassociated with definitive endoderm (FOXA2, SOX17, CER1, and CXCR4),when compared with control samples not treated with IGF (FIG. 6).Similar results were observed with extraembryonic markers (SOX7, AFP).Addition of 50-200 ng/ml of IGF did increase expression of the embryonicmarker NANOG.

Cultures treated with medium supplemented with FBS were much moresensitive to the inhibitory effect of IGF. In these cultures, theexpression of SOX17, HNF3B and CXCR4 decreased with increasingconcentration of IGF. These observations was further supported by immunofluorescence (IF) staining for the DE marker SOX17 (Catalogue #AF 1924,R & D systems, MN) (FIGS. 8-9).

As summarized in Table III, only at super physiological concentrationsof IGF (50-200 ng/ml) in BSA treated cultures there was a drop inexpression of CXCR4+CD9− cells and an increase in expression ofCXCR4−CD9+ fraction. However, with increasing doses of IGF, FBS treatedcultures showed a more significant drop in expression of CXCr4+CD9−fraction as compared to BSA treated cultures. The above examplescollectively show that in the absence of FBS, physiologicalconcentrations of IGF or insulin are not inhibitory to induction of DEmarkers.

TABLE III Treatment % CXCR4 + CD9− % CXCR4 − CD9+ % CXCR4 − CD9− BSA 920.9 2.6 FBS 93 2.7 5.9 BSA + 1 92 0.8 3 ng/ml IGF FBS + 1 89 3 3.6 ng/mlIGF BSA + 10 90 1.3 5.3 ng/ml IGF FBS + 10 87 6.4 3.3 ng/ml IGF BSA + 5087 6 3.5 ng/ml IGF FBS + 50 78 6.8 13.2 ng/ml IGF BSA + 200 79 10 8ng/ml IGF FBS + 200 70 13.4 12.9 ng/ml IGF

Example 4 IGF Concentrations in Various Lots of FBS

An IGF-1 Kit was purchased from Diagnostic Systems Laboratories (DSL)(Cat. DSL-10-2800) and was used for the detection. Twenty micro liters(20 μl) of serum (duplicates) were pre-treated and then 20 μl of dilutedsample was used for assay. For medium samples, 20 μl of samples weredirectly used for assay. The assay was performed following theinstruction provided by the kit. This kit can detect both human andbovine IGF-1 as the 2 monoclonal antibodies used for the kit are againstto the homolog peptide sequences.

Test samples: The following test samples were used:

4A/5A: Hyclone Newborn Calf Serum; Lot AKM12868

4B/5B: NIH-FBS (from aliquot@ −20 C)

4C/5C: Hyclone FBS, Lot: ATK33398

4D/5D: Hyclone FBS, Lot: AUK 54924

4E/5E: Human serum, Lot: A70184, from Valley Biomedical Inc.

4F/5F: Knockout Serum; Invitrogen, Lot: 557914

4F/5F: F12 DMEM, Invitrogen, Lot: 692281

4H/5H: MEF Condition Medium, Lot: 011410 (Day 5)

Control Samples: The following control samples were used:

Background: “0” IGF-1 (negative control, from kit); F12 sample listedabove is also serves as a negative control.

2 positive controls (127 ng/ml and 241 ng/ml; from the kit).

Results: The sensitivity of the assay for serum was greater than 10ng/ml; and for medium was greater than 0.1 ng/ml.

Concentration Known positives determined by the (ng/ml) Assay (ng/ml) SE127 ng/ml 126.8 8.2 241 ng/ml 233.8 4.2

IGF-1 Duplicates Samples (ng/ml) SE 4A/5A Hyclone Newborn Calf Serum;Lot AKM12868 29.78 0.41 4B/5B NIH-FBS (from aliquot@ −20 C.) 49.16 2.684C/5C Hyclone FBS, Lot: ATK33398 80.29 5.39 4D/5D Hyclone FBS, Lot: AUK54924 76.45 1.99 4E/5E Human serum, Lot: A70184, from Valley 55.65 2.28Biomedical Inc. 4F/5F Knockout Serum; Invitrogen, Lot: 557914 12.07 0.154G/5G F12 DMEM, Invitrogen, Lot: 692281 ND* ND 4H/5H MEF-ConditionMedium, Lot: 011410 (Day 5)  1.33** 0.07

Example 5 Role of Insulin/IGF and FBS in the Differentiation of HumanPluripotent Stem Cells to Cells Expressing Markers Characteristic of theDefinitive Endoderm Lineage: Single Cell Seeding

Cells of the human embryonic stem cells line H1 (p40-p52) were seeded assingle cells at a density of 100000 cells/cm² on MATRIGEL® (1:30dilution) (BD Biosciences; Cat #356231)-coated dishes in MEF-CM (mouseembryonic fibroblast conditioned media) supplemented with 16 ng/ml ofFGF2 (Catalog#100-18B, PeproTech, NJ) and 10 μM of Y27632 (Rockinhibitor, Catalogue#Y0503, Sigma, Mo.). 72 hrs post seeding, cultureswere differentiated into definitive endoderm (DE) as follows:

-   -   a. with MCDB-131 (Catalogue#10372-019, Invitrogen, CA) medium        supplemented with 0.2% FBS (Catalogue#SH30070.03, Hyclone, UT),        0.0025 g/ml sodium bicarbonate (Catalogue #S3187, Sigma, Mo.),        1× GlutaMax™ (Catalogue #35050-079, Invitrogen, Ca) and 100        ng/ml GDF8 (Catalogue#120-00, PeproTech, NJ) plus 2.5 μM of the        GSK3B inhibitor        14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜0.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one        for one day, then    -   b. MCDB-131 medium supplemented with FBS, sodium bicarbonate,        Glutamax, and 100 ng/ml GDF8 for an additional three days.

0.5% FBS was used for day 2 and 2% FBS was used for days 3-4. Besidesregular FBS, heat treated FBS (Catalogue#F4135, Sigma, Mo.) and charcoalstripped treated FBS (Catalogue#F6765, Sigma, Mo.) were also tested.Some of the FBS treated cultures were also treated with variousconcentrations of IGF (10-100 ng/ml) or insulin (10-100 ng/ml).

At day 4, samples were collected for analysis by FACS and real-time PCR.In contrast to BSA treated cultures (see previous Examples) in thepresence of FBS, addition of insulin or IGF dose dependently downregulated markers associated with definitive endoderm such as SOX17 andCCXR4. See FIG. 9. The expression of the pluripotency marker NANOG wasalso upregulated.

Publications cited throughout this document are hereby incorporated byreference in their entirety. Although the various aspects of theinvention have been illustrated above by reference to examples andpreferred embodiments, it will be appreciated that the scope of theinvention is defined not by the foregoing description but by thefollowing claims properly construed under principles of patent law.

What is claimed is:
 1. A population of cells, wherein greater than 85%of the cells in the population express markers characteristic of thedefinitive endoderm lineage.
 2. A method for generating a population ofcells wherein greater than 85% of the cells in the population expressmarkers characteristic of the definitive endoderm lineage, comprisingthe steps of: a. Culturing a population of pluripotent stem cells, b.Differentiating the population of pluripotent stem cells to a populationof cells wherein greater than 85% of the cells in the population expressmarkers characteristic of the definitive endoderm lineage in mediumlacking serum and supplemented with BSA and a factor selected from thegroup consisting of insulin and IGF-1.
 3. The method of claim 2, whereinthe population of pluripotent stem cells are differentiated to apopulation of cells wherein greater than 85% of the cells in thepopulation express markers characteristic of the definitive endodermlineage using activin A and a Wnt ligand.
 4. The method of claim 2,wherein the population of pluripotent stem cells are differentiated to apopulation of cells wherein greater than 85% of the cells in thepopulation express markers characteristic of the definitive endodermlineage using GDF-8 and at least one other factor is selected from thegroup consisting of: an aniline-pyridinotriazine, a cyclicaniline-pyridinotriazine,N-{[1-(Phenylmethyl)azepan-4-yl]methyl}-2-pyridin-3-ylacetamide,4-{[4-(4-{[2-(Pyridin-2-ylamino)ethyl]amino}-1,3,5-triazin-2-yl)pyridin-2-yl]oxy}butan-1-ol,3-({3-[4-({2-[Methyl(pyridin-2-yl)amino]ethyl}amino)-1,3,5-triazin-2-yl]pyridin-2-yl}amino)propan-1-ol,N˜4˜-[2-(3-Fluorophenyl)ethyl]-N˜2˜-[3-(4-methylpiperazin-1-yl)propyl]pyrido[2,3-d]pyrimidine-2,4-diamine,1-Methyl-N-[(4-pyridin-3-yl-2-{[3-(trifluoromethyl)phenyl]amino}-1,3-thiazol-5-yl)methyl]piperidine-4-carboxamide,1,1-Dimethylethyl{2-[4-({5-[3-(3-hydroxypropyl)phenyl]-4H-1,2,4-triazol-3-yl}amino)phenyl]ethyl}carbamate,1,1-Dimethylethyl{[3-({5-[5-(3-hydroxypropyl)-2-(methyloxy)phenyl]-1,3-oxazol-2-yl}amino)phenyl]methyl}carbamate,1-({5-[6-({4-[(4-Methylpiperazin-1-yl)sulfonyl]phenyl}amino)pyrazin-2-yl]thiophen-2-yl}methyl)piperidin-4-ol,1-({4-[6-({4-[(4-Methylpiperazin-1-yl)sulfonyl]phenyl}amino)pyrazin-2-yl]thiophen-2-yl}methyl)piperidine-4-carboxamide,and2-{[4-(1-Methylethyl)phenyl]amino}-N-(2-thiophen-2-ylethyl)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxamide.5. The method of claim 4, wherein the at least one other factor is14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6˜0.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one.6. The method of claim 2, wherein the population of pluripotent stemcells is differentiated in the medium lacking serum and supplementedwith BSA and a factor selected from the group consisting of insulin andIGF-1 for a period of at least 6 days.
 7. The method of claim 2, whereinthe population of pluripotent stem cells is differentiated in the mediumlacking serum and supplemented with BSA and a factor selected from thegroup consisting of insulin and IGF-1 for a period of at least 7 days.